Pharmaceutical composition for dermatology and uses thereof

ABSTRACT

The present invention refers to a composition comprising a conditioned cell culture medium obtained or obtainable by a process which comprises culturing a population of mesenchymal stromal cells (MSCs), in which at least about 25%, at least about 30%, at least about 35%, at least about 40%, at least about 45%, at least about 50%, at least about 55%, at least about 60%, at least about 65%, at least about 70%, at least about 75%, at least about 80%, at least about 85%, at least about 90%, at least about 95%, at least about 96%, at least about 97%, at least about 98%, or at least about 99%, of said population by number of cells are MSCs obtained from a mammal pertaining to the genus Canis, in a nutrient rich liquid prepared for cell culture, preferably a basal media; and collecting the conditioned cell culture medium, wherein preferably the nutrient rich liquid is an animal/human serum-free media or a chemically defined animal/human serum-free and xeno free media designed to grow MSCs, for use in therapy.

FIELD OF THE INVENTION

The present invention relates to the fields of development, cellbiology, molecular biology and genetics. More particularly, theinvention relates to a method of deriving a conditioned media frommesenchymal stem cells obtained from a mammal pertaining to the genusCanis and uses of such media for the treatment of skin inflammatorydisorders such as psoriasis or atopic dermatitis.

BACKGROUND OF THE INVENTION

The skin, once thought to be immunologically sequestered from the body,is now deemed to be an integral part of a multisystem inflammatory axis.

Atopic dermatitis (AD) and psoriasis, especially in their most severeforms, have been linked to a variety of systemic inflammatory disordersand comorbidities. These comorbidities highlight that inflammatory skindiseases of childhood are serious chronic multisystem illnesses and notmerely cosmetic conditions.

Atopic dermatitis comorbidities vary by age and length of illness. Someof the most common comorbidities are included in the gold-standard ADdiagnostic criteria by Hanifin and Rajka (Acta Derm Venereol Suppl(Stockh) 1980; 92:44-47). These criteria include pruritus, chronic orrecurrent dermatitis, specific distribution by age (e.g., flexuraldisease of childhood) and personal and/or family history of atopy, i.e.,food allergies, asthma and allergic rhinoconjunctivitis. Minor featuresinclude more than a dozen comorbid conditions, including ichthyosisvulgaris; bacterial and viral infections; allergic predisposition withpositive immunoglobulin E and skin prick testing; eye findings such ascataracts; and excessive skin reactivity to touching foods, pressure andenvironmental triggers. These major and minor criteria actually havebeen staring us in the face for almost 40 years with the concept ofcomorbidities being part and parcel of the definition of atopicdermatitis and the atopic diathesis.

Recently, both pediatric psoriasis and atopic dermatitis have beenlinked to cutaneous infections and psychosocial disorders such asanxiety, depression and hyperactivity for the children involved andtheir parents. Linkage to cutaneous autoimmunity, including vitiligo andalopecia areata, may be noted in both sets of diseases.

The most important and well-described series of comorbidities shared bythese two diseases is the association with obesity and the metabolicsyndrome—a cluster of conditions characterized by increased risk ofheart disease, stroke and diabetes. Early childhood obesity has beenassociated with atopic dermatitis development and severity. Inpsoriasis, increased abdominal girth and obesity may precede disease bya few years, suggesting their role as triggers in disease. Oneinteresting feature of psoriatic disease is promising data from adultswho had weight loss surgery and experienced psoriatic diseaseimprovement.

Despite many common comorbidities, paediatric atopic dermatitis andpaediatric psoriasis have many distinctive features.

In atopic dermatitis, the skin barrier is both weak and weakened byinflammation, allowing a series of unusual allergic features, i.e., theatopic march and infectious complications. Alternatively, in psoriasisthe skin is triggered to thicken, and the joints may become inflamed.

Further divergence is seen in the clinical manifestations. In psoriasis,arthritis is the leading comorbidity, sometimes triggered bystreptococcal disease. There also is a far more definitive associationwith metabolic syndrome features such as hypertension, hyperlipidemiaand insulin resistance. Despite the common nature of psoriasis and thefrequency of disease, atopic dermatitis has developed a more extensivelaundry list of comorbidities, including infantile seborrheicdermatitis, Malassezia sensitization, dust allergy, asthma, foodallergy, environmental allergens, contact dermatitis (e.g., lanolin,fragrance), prurigo, sleep disturbance, upper respiratory infections,warts, coxsackie generalization (e.g., eczema coxsackium) and cataracts.

At any rate, the development of screening tools and effectivepharmacological interventions for patients of any age with inflammatoryskin disease such as psoriasis or atopic dermatitis is a work inprogress and it has become a crucial need nowadays. In this sense, thepresent invention provides for the use of conditioned media in whichMSCs (mesenchymal stem cells) derived from a mammal pertaining to thegenus Canis are cultured in order to obtain such conditioned mediasuitable for the treatment of skin inflammatory disorders such aspsoriasis or atopic dermatitis. It is particularly important tohighlight, that the composition disclosed in the present invention isparticularly effective in the xenogeneic treatment of human atopicdermatitis even though there is a high phylogenetic divergence betweenhumans and dogs (see FIGS. 7 to 9).

In this regard, WO2008155659 discloses compositions for preventing ortreating skin defects comprising conditioned cell medium frommesenchymal stem cells (MSC). In addition, this document indicates thatMSCs can be obtained from humans, pigs, dogs, cats, mice, horses andother mammals. However, this document fails to specifically refer to“atopic dermatitis” or “psoriasis” and much less that these diseases canbe treated with compositions comprising a conditioned cell medium frommesenchymal stem cells (MSC) obtained from dogs. In this sense, example2 of the present invention indicates that MSCs from different originsprovide different conditioned media under a qualitative and quantitativepoint of view. In fact, as illustrated in the figures (FIG. 2 incomparison to FIGS. 1, 3 and 4), a conditioned culture media from dogadipose MSCs is considerably different from a conditioned culture mediaobtained from human, cat or horse adipose tissue. Again, WO2008155659provides no indication that a conditioned culture media specificallyobtained from dog adipose MSCs is particularly useful for an effectivetreatment of dermatitis such as atopic dermatitis and psoriaris in humanbeings.

In addition, the fact that the present invention is preferably focus inthe xenogeneic treatment of human atopic dermatitis, is certainlycounterintuitive in light of documents such as WO2017041133 wherein thefollowing statements can be found: “Adipose tissue may be human adiposetissue or mammalian animal adipose tissue, such as canine, equine orfeline. Typically the source of the adipose tissue will be of the samespecies as the intended recipient of the MSCs . . . ”

Furthermore, although Mohd Matin Ansari “Therapeutic potential of caninebone marrow derived mesenchymal stem cells and its conditioned media indiabetic rat wound healing”, Journal of stem cell research & therapy,vol. 3, no. 3, 1 Jan. 2013 (2014-01-01), XP055406815, discloses thepotential effectiveness of canine cells or its conditioned media in thetreatment of wounds in different species such as in diabetic rats, suchwounds are made by making a full thickness excision in the skin of therat. On the contrary, skin inflammation causing atopic dermatitis rashis considered a type of allergic response. Therefore, the treatment ofwounds generated in Mohd Matin Ansari et al and the treatment of atopicdermatitis are completely unrelated.

Finally, WO2017144552 is explicitly directed to compositions thatmandatorily comprise dimethyl sulfoxide (DMSO), in fact DMSO is anessential feature in WO2017144552 since it enhances the treatment of thediseases detailed therein. However, WO2017144552 is not directed to thexenogeneic treatment of human atopic dermatitis or psoriaris,particularly in the context of a DMSO free composition, by usingconditioned cell culture mediums obtained or obtainable by a processwhich comprises culturing a population of mesenchymal stromal cells(MSCs), wherein at least 50% of said population by number of cells areMSCs obtained from a mammal pertaining to the genus Canis.

Therefore, the present invention is, as far as we know, the first toprovide a composition comprising a conditioned cell culture mediumobtained or obtainable by a process which comprises culturing apopulation of mesenchymal stromal cells (MSCs), wherein at least 50% ofsaid population by number of cells are MSCs obtained from a mammalpertaining to the genus Canis, for use in the xenogeneic treatment ofhuman atopic dermatitis.

BRIEF DESCRIPTION OF THE INVENTION

According to the invention, instead of using stem cells, injured or losttissues may be regenerated or repaired through enhancement of endogenoustissue repair by applying secretions from MSCs instead of, or inaddition to, MSCs themselves. Specifically, we provide for the use ofconditioned media in which the MSCs derived from a mammal pertaining tothe genus Canis are cultured in order to obtain such conditioned mediasuitable for the treatment of skin inflammatory disorders such aspsoriasis or atopic dermatitis.

In particular, in table II (see description) we show a qualitative andquantitative comparison of the polypeptides secreted by MSCs from amammal pertaining to the genus Canis and those secreted from othermammals such as MSCs from cats or humans. In addition, the examplesdisclosed in the present specification clearly show a therapeutic effectlinked to the use of the conditioned media obtained from culturing MSCsderived from a mammal pertaining to the genus Canis, in particular atherapeutic effect linked to the treatment of atopic dermatitis orpsoriasis.

With this approach, the present confounding issues associated with cellbased therapy i.e. immune compatibility, tumorigenicity, xenozooticinfections, costs, and waiting time if autologous cell preparations areused will be eliminated. Such an approach could potentially provide forthe development of “off-the-shelf” MSC-based therapeutics at affordablecosts and with better quality control and consistency.

The practice of the present invention will employ, unless otherwiseindicated, conventional techniques of chemistry, molecular biology,microbiology, recombinant DNA and immunology, which are within thecapabilities of a person of ordinary skill in the art. Such techniquesare explained in the literature. See, for example, J. Sambrook, E. F.Fritsch, and T. Maniatis, 1989, Molecular Cloning: A Laboratory Manual,Second Edition, Books 1-3, Cold Spring Harbor Laboratory Press; Ausubel,F. M. et al. (1995 and periodic supplements; Current Protocols inMolecular Biology, ch. 9, 13, and 16, John Wiley & Sons, New York, N.Y.); B. Roe, J. Crabtree, and A. Kahn, 1996, DNA Isolation andSequencing: Essential Techniques, John Wiley & Sons; J. M. Polak andJames O'D. McGee, 1990, Oligonucleotide Synthesis: A Practical Approach,Irl Press; D. M. J. Lilley and J. E. Dahlberg, 1992, Methods ofEnzymology: DNA Structure Part A: Synthesis and Physical Analysis of DNAMethods in Enzymology, Academic Press; Using Antibodies: A LaboratoryManual: Portable Protocol NO. I by Edward Harlow, David Lane, Ed Harlow(1999, Cold Spring Harbor Laboratory Press, ISBN 0-87969-544-7);Antibodies: A Laboratory Manual by Ed Harlow (Editor), David Lane(Editor) (1988, Cold Spring Harbor Laboratory Press, ISBN0-87969-314-2), 1855; and Lab Ref: A Handbook of Recipes, Reagents, andOther Reference Tools for Use at the Bench, Edited Jane Roskams andLinda Rodgers, 2002, Cold Spring Harbor Laboratory, ISBN 0-87969-630-3.Each of these general texts is herein incorporated by reference.

Thus a first aspect of the invention refers to a composition comprisinga conditioned cell culture medium obtained or obtainable by a processwhich comprises culturing a population of mesenchymal stromal cells(MSCs), in which at least about 25%, at least about 30%, at least about35%, at least about 40%, at least about 45%, at least about 50%, atleast about 55%, at least about 60%, at least about 65%, at least about70%, at least about 75%, at least about 80%, at least about 85%, atleast about 90%, at least about 95%, at least about 96%, at least about97%, at least about 98%, or at least about 99%, of said population bynumber of cells are MSCs obtained from a mammal pertaining to the genusCanis or immortalized mesenchymal stromal cells obtained therefrom, in anutrient rich liquid prepared for cell culture, preferably a basalmedia, such as DMEM, MEM, RPMI, or HAM'S with or without supplementssuch as serum containing media, serum free media, protein free media andchemically defined media; and collecting the conditioned cell culturemedium,

wherein preferably the nutrient rich liquid is an animal and humanserum-free media designed to grow MSCs, andwherein preferably said composition does not comprise dimethyl sulfoxide(DMSO),for use in therapy.

It is preferably noted, that preferably the composition of the firstaspect of the invention as a whole does not comprise human and animalserum components.

In a preferred embodiment of the first aspect of the invention or of anyof its preferred embodiments, the MSCs are obtained from a mammalpertaining to the dog species.

In another preferred embodiment of the first aspect of the invention orof any of its preferred embodiments, the mesenchymal stromal cells areumbilical-cord derived stromal cells, adipose tissue-derived stromalcells, expanded mesenchymal stromal cells, expanded adiposetissue-derived stromal cells, bone-marrow derived stromal cells,expanded bone-marrow derived stromal cells or immortalized mesenchymalstromal cells obtained from any of the afore mentioned sources.

In another preferred embodiment of the first aspect of the invention orof any of its preferred embodiments, the nutrient rich liquid preparedfor cell culture is a buffered saline solution comprising amino acidsand vitamins supplemented with sodium pyruvate and glutamine.Preferably, the nutrient rich liquid prepared for cell culture is thebasal media DMEM, MEM, RPMI, or HAM'S with or without supplements suchas serum containing media, serum free media, protein free media andchemically defined media.

In a second aspect of the invention, the medical composition of thefirst aspect of the invention is used in the treatment or prevention ofdermatitis in a human or animal subject. Preferably, said dermatitis isselected from the list consisting of atopic dermatitis, eczema andpsoriasis. More preferably, the medical composition of the first aspectof the invention is use in the xenogeneic treatment of atopicdermatitis, eczema and/or psoriasis in a human subject.

A third aspect of the invention refers to the medical composition of thefirst aspect of the invention or to the composition for use according tothe second aspect of the invention, wherein such composition isconfigurable to deliver the required amount of conditioned medium at theappropriate interval, in order to achieve optimal treatment.

In a preferred embodiment of the third aspect of the invention, suchcomposition comprises excipients. Preferred excipients are thosesuitable for being included in topical compositions (i.e. dermatologicalacceptable excipient). The following auxiliary agents mentioned belowcan independent of each other be present in such composition: gellingagents, oils, waxes, thickening agents, hydrophilic or hydrophobicpolymers, emulsifying agents, emollients, fatty acids, organic solvents,antioxidants, stabilizers, sequestering agents, acidifying or basifyingagents, emulsifiers, emollients, surfactants, film formers, biologicaladditives to enhance performance and/or consumer appeal such as aminoacids, proteins, vanilla, aloe extract or bioflavinoids, bufferingagents, chelating agents such as ethylenediaminetetra-acetic acid (EDTA)or oxalic acid, colorants, dyes, propellants, antifoaming agents,wetting agents, vitamins, emulsion stabilizers, pH adjusters, thickeningagents, fragrances, preservatives, opacifying agents, water and/oralcohols. The aforementioned auxiliary agents for the composition areused in the usual amounts known by those skilled in the art. Suitableoils for the compositions are selected from animal or vegetable orsynthetic oils. Particularly preferred oils are selected from the groupcomprising liquid petrolatum, liquid paraffin, volatile and non-volatilesilicone oils, isoparaffins, polyalphaolefins, fluorated andperfluorated oils. Suitable stabilizers for the compositions of theinvention can be of non-ionic, anionic, cationic and amphiphilic nature.Preferred stabilizers are selected from the group comprisingpolyethylenglycol (PEG) and derivatives thereof, tweens, tritons, spans,polygycerines, polyalkyl glycerides, alkyl sulfonates, aryl sulfonates,alkyl phosphates, derivatives of alkyl-betaine andphosphatidylglycerole.

In another preferred embodiment of the third aspect of the invention,the composition is suitable for administering to a subject, preferablyto a human subject, by any of the following routes of administration:intravenous, oral and topical. In a preferred embodiment of the thirdaspect of the invention the composition of the invention is a topicalformulation that may be formulated in liquid or in semi-solid form,preferably as liquid, fluid, foam, cream, gel, paste, balsam, spray,ointment, lotion, conditioner, tonic, milk, mousse, emulsion, serum,oil, stick, shampoo, jelly, suspension, dispersion, lacquer, paint,elixir, drop or aerosol. In a particular embodiment, the activecompounds of the invention were administered topically in a liposomalpreparation.

BRIEF DESCRIPTION OF THE FIGURES

FIG. 1. Representative image of a Human XL Cytokine Array coordinates,hybridated with conditioned cultured media from human adipose derivedmesenchymal stem cells.

FIG. 2. Representative image of a Human XL Cytokine Array coordinates,hybridated with conditioned cultured media from dog adipose derivedmesenchymal stem cells.

FIG. 3. Representative image of a Human XL Cytokine Array coordinates,hybridated with conditioned cultured media from cat adipose derivedmesenchymal stem cells.

FIG. 4. Representative image of a Human XL Cytokine Array coordinates,hybridated with conditioned cultured media from horse adipose derivedmesenchymal stem cells.

FIG. 5. Pekingnese dog treated with dog conditioned media. Images showthe evolution of the wounds at four different points in time. Rush andskin peeling decreases during treatment up to complete remission in the6^(th) week.

FIG. 6. Siamese cat treated with dog conditioned media. Images show theevolution of the wound at the beginning and the end of the treatment atweek number 5. The size of the wound decreased as is shown in thecomparative superposition.

FIG. 7. 79 years old human male treated with dog conditioned media.Images show the evolution of the psoriatic wounds during the 35 daysthat last the treatment in leg and hands. Peeling and scales diminishnotably from the skin.

FIG. 8. 37 years old female treated with dog conditioned media. Imagesshow the evolution of the psoriatic wounds during the 21 days that lastthe treatment in arm and foot. Peeling and scales diminish notably fromthe skin.

FIG. 9. 58 years old human male treated with dog conditioned media.Images show the evolution of the psoriatic wounds during the 36 daysthat last the treatment in leg and hand. Peeling and scales disappearfrom the skin.

DETAILED DESCRIPTION OF THE INVENTION Definitions

As used herein, the following terms and phrases shall have the meaningsset forth below. Unless defined otherwise, all technical and scientificterms used herein have the same meaning as commonly understood to one ofordinary skill in the art to which this invention belongs.

The articles “a” and “an” refer to one or to more than one (i.e., to atleast one) of the grammatical object of the article. By way of example,“an element” means one element or more than one element.

The term “about” when used in relation to a value relates to thevalue±10%.

By “adipose tissue” is meant any fat tissue. The adipose tissue may bebrown or white adipose tissue, derived from, for example, subcutaneous,omental/visceral, mammary, gonadal, or other adipose tissue site.Preferably, the adipose tissue is subcutaneous white adipose tissue. Theadipose tissue may comprise a primary cell culture or an immortalizedcell line. The adipose tissue may be from any organism having fattissue. In some embodiments, the adipose tissue is mammalian, and infurther embodiments the adipose tissue is human. A convenient source ofadipose tissue is liposuction surgery. However, it will be understoodthat neither the source of adipose tissue nor the method of isolation ofadipose tissue is critical to the invention. If cells as describedherein are desired for autologous transplantation into a subject, theadipose tissue will be isolated from that subject.

“Adipose tissue-derived stromal cells (ASCs)” refers to MSCs thatoriginate from adipose tissue, generally from human adipose tissue(hASCs). These cells have been used in the present invention toreproduce the invention.

The term “cells/kg” as used herein shall be taken to mean the number ofcells (e.g. MSC) administered per kilogram of patient body weight.

The term “culture” refers to the growth of cells, organisms,multicellular entities, or tissue in a medium. The term “culturing”refers to any method of achieving such growth, and may comprise multiplesteps. The term “further culturing” refers to culturing a cell,organism, multicellular entity, or tissue to a certain stage of growth,then using another culturing method to bring said cell, organism,multicellular entity, or tissue to another stage of growth. A “cellculture” refers to a growth of cells in vitro. In such a culture, thecells proliferate, but they do not organize into tissue per se. A“tissue culture” refers to the maintenance or growth of tissue, e.g.,explants of organ primordial or of an adult organ in vitro so as topreserve its architecture and function. A “monolayer culture” refers toa culture in which cells multiply in a suitable medium while mainlyattached to each other and to a substrate. Furthermore, a “suspensionculture” refers to a culture in which cells multiply while suspended ina suitable medium. Likewise, a “continuous flow culture” refers to thecultivation of cells or explants in a continuous flow of fresh medium tomaintain cell growth, e.g. viability. The term “conditioned media”refers to the supernatant, e.g. free of the cultured cells/tissue,resulting after a period of time in contact with the cultured cells suchthat the media has been altered to include certain paracrine and/orautocrine factors produced by the cells and secreted into the culture. A“confluent culture” is a cell culture in which all the cells are incontact and thus the entire surface of the culture vessel is covered,and implies that the cells have also reached their maximum density,though confluence does not necessarily mean that division will cease orthat the population will not increase in size.

The term “culture medium” or “medium” is recognized in the art, andrefers generally to any substance or preparation used for thecultivation of living cells. The term “medium”, as used in reference toa cell culture, includes the components of the environment surroundingthe cells. Media may be solid, liquid, gaseous or a mixture of phasesand materials. Media include liquid growth media as well as liquid mediathat do not sustain cell growth. Media also include gelatinous mediasuch as agar, agarose, gelatine and collagen matrices. Exemplary gaseousmedia include the gaseous phase that cells growing on a petri dish orother solid or semisolid support are exposed to. The term “medium” alsorefers to material that is intended for use in a cell culture, even ifit has not yet been contacted with cells. In other words, a nutrientrich liquid prepared for bacterial culture is a medium. Similarly, apowder mixture that when mixed with water or other liquid becomessuitable for cell culture may be termed a “powdered medium”. “Definedmedium” refers to media that are made of chemically defined (usuallypurified) components. “Defined media” do not contain poorlycharacterized biological extracts such as yeast extract and beef broth.“Rich medium” includes media that are designed to support growth of mostor all viable forms of a particular species. Rich media often includecomplex biological extracts. The term “basal medium” refers to a mediumwhich promotes the growth of many types of microorganisms which do notrequire any special nutrient supplements. Most basal media generallycomprise four basic chemical groups: amino acids, carbohydrates,inorganic salts, and vitamins. A basal medium generally serves as thebasis for a more complex medium, to which supplements such as serum,buffers, growth factors, lipids, and the like are added. Examples ofbasal media include, but are not limited to, Eagles Basal Medium,Minimum Essential Medium, Dulbecco's Modified Eagle's Medium, Medium199, Nutrient Mixtures Ham's F-10 and Ham's F-12, McCoy's 5 A,Dulbecco's MEM/F-I 2, RPMI 1640, and Iscove's Modified Dulbecco'sMedium. It is noted that many modifications of Eagle's Medium have beendeveloped since the original formulation appeared in the literature.Among the most widely used of these modifications is Dulbecco's ModifiedEagle's medium (DMEM). DMEM is a modification of Basal Medium Eagle(BME) that contains a four-fold higher concentration of amino acids andvitamins, as well as additional supplementary components. The originalDMEM formula contains 1000 mg/L of glucose and was first reported forculturing embryonic mouse cells (low glucose DMEM). A further alterationwith 4500 mg/L glucose has proved to be optimal for cultivation ofcertain cell types (high glucose DMEM). DMEM is a preferred basal mediain the context of the present invention. It is noted that the basalmedia referred to in the present invention is an animal/human serum-freemedia or a chemically defined serum-free and xeno free media designed togrow MSCs such as Eagles Basal Medium or Dulbecco's Modified Eagle'sMedium (DMEM).

In the context of the present invention, an animal and human serum-freemedia is understood as a media that contains no animal or human-derivedblood or serum components. In particular, it refers to a media thatcontains no human or animal proteins present in the blood of humans oranimals such as albumin or fetal bovine serum or fetal calf serum (thatis to say, the medium contains no animal or human-derived blood or serumcomponents). An example of an animal and human serum-free media is DMEM.

The terms “comprise” and “comprising” are used in the inclusive, opensense, meaning that additional elements may be included.

The term “expanded” as used herein when referring to cells shall betaken to have its usual meaning in the art, namely cells that have beenproliferated in vitro. A MSC can be expanded to provide a population ofcells that retain at least one biological function of the MSC, typicallythe ability to adhere to a plastic surface, under standard cultureconditions. The expanded population of cells may retain the ability todifferentiate into one or more cell types.

The term “including” is used herein to mean “including but not limitedto”. “Including” and “including but not limited to” are usedinterchangeably.

“Mesenchymal stromal cells (also referred to herein as “MSCs”) aremultipotent stromal cells, i.e. they are cells which are capable ofgiving rise to multiple different types of cells. These types of cellsare defined by their plastic adherent growth and subsequent expansionunder specific culture conditions and by their in vitro and in vivodifferentiation potential (Caplan A I: Mesenchymal Stem Cells. J OrthopRes 1991, 9:641-650; Zuk P A, Zhu M, Mizuno H, Huang J, Futrell J W,Katz A J, Benhaim A J, Lorenz H P, Hedrick M H: Multi-lineage cells fromhuman adipose tissue: implication for cell-based therapies. Tissue Eng2001, 7:211-228; Huang J I, Beanes S R, Zhu M, Lorenz H P, Hedrick M H,Benhaim P: Rat extramedullary adipose tissue as a source ofosteochondrogenic progenitor cells. Plast Reconstr Surg 2002,109:1033-1041; Dennis J E, Caplan A I: Differentiation potential ofconditionally immortalized mesenchymal progenitor cells from adultmarrow of a H-2Kb-tsA58 transgenic mouse. J Cell Physiol 1996,167(3):523-538; Johnstone B, Hering T_(M), Caplan A I, Goldberg V M, YooJ U: In vitro chondrogenesis of bone marrow-derived mesenchymalprogenitor cells. Exp Cell Res 1998, 238(1):265-272; Dennis J E, MerriamA, Awadallah A, Yoo J U, Johnstone B, Caplan A I: A quadr-icpotentialmesenchymal progenitor cell isolated from the marrow of an adult mouse.J Bone Miner Res 1999, 14(5):700-709; Kopen G C, Prockop D J, Phinney DG: Marrow stromal cells migrate throughout forebrain and cerebellum, andthey differentiate into astrocytes after injection into neonatal mousebrains. Proc Natl Acad Sci USA 1999, 96:10711-10716; Toma C, Pittenger MF, Cahill K S, Byrne B J, Kessler P D: Human mesenchymal stem cellsdifferentiate to a cardiomyocyte phenotype in the adult murine heart.Circulation 2002, 105:93-98). MSCs are well known in the literature asshown above and as such form part of the common general knowledgeattributable to the skilled person in the art.

MSCs have been isolated from numerous mammals in order to practice thepresent invention. For example, MSCs have been derived from variousequine tissues, including bone marrow (Arnhold S J, Goletz I, Klein H,Stumpf G, Beluche L A, Rohde C, Addicks K, Litzke L F. Isolation andcharacterization of bone marrow-derived equine mesenchymal stem cells.Am J Vet Res 2007, 68, 1095-1105; Kisiday J D, Kopesky P W, Evans C H,Grodzinsky A J, Mcllwraith C W, Frisbie D D. Evaluation of adult equinebone marrow- and adipose-derived progenitor cell chondrogenesis inhydrogel cultures. J Orthop Res 2008, 26, 322-33; Smith R K W, Korda M,Blunn G W, Goodship A E. Isolation and implantation of autologous equinemesenchymal stem cells from bone marrow into the superficial digitalflexor tendon as a potential novel treatment. Equine Vet J 2003, 35,99-102), adipose tissue (Vidal M A, Kilroy G E, Johnson J R, Lopez M J,Moore R M, Gimble J M. Cell growth characteristics and differentiationfrequency of adherent equine bone marrow-derived mesenchymal stromalcells: adipogenic and osteogenic capacity. Vet Surg 2006, 35, 601-610),peripheral blood, umbilical cord blood, umbilical cord matrix, andamniotic fluid. However, it is noted that the present invention isdirected to the use of canine MSCs, in particular isolated from bonemarrow and adipose tissue. Such canine MSCs are well documented inHiroshi Takemitsu et al. Comparison of bone marrow and adiposetissue-derived canine mesenchymal stem cells. BMC Veterinary Research2012, 8:150. Lastly, MSCs have been successfully isolated from bonemarrow and adipose tissue (Caplan A I: Mesenchymal Stem Cells. J OrthopRes 1991, 9:641-650. Zuk P A, Zhu M, Mizuno H, Huang J, Futrell J W,Katz A J, Benhaim A J, Lorenz H P, Hedrick M H: Multi-lineage cells fromhuman adipose tissue: implication for cell-based therapies. Tissue Eng2001, 7:211-228. Huang J I, Beanes S R, Zhu M, Lorenz H P, Hedrick M H,Benhaim P: Rat extramedullary adipose tissue as a source ofosteochondrogenic progenitor cells. Plast Reconstr Surg 2002,109:1033-1041) in humans.

A “patient”, “subject” or “host” to be treated by the subject method maymean either a human or non-human animal.

The term “pharmaceutical composition” refers to a composition intendedfor use in therapy, no matter if it is for human or animal therapy, sothat within the term pharmaceutical composition of the present inventionveterinary compositions are encompassed. The compositions of theinvention are pharmaceutical compositions, intended for use in thetreatment of a patient or host.

The phrase “pharmaceutically acceptable” is employed herein to refer tothose compounds, materials, compositions, and/or dosage forms which are,within the scope of sound medical judgment, suitable for use in contactwith the tissues of human beings and animals without excessive toxicity,irritation, allergic response, or other problem or complication,commensurate with a reasonable benefit/risk ratio.

The phrase “pharmaceutically acceptable carrier” as used herein means apharmaceutically acceptable material, composition or vehicle, such as aliquid or solid filler, diluent, excipient, or solvent encapsulatingmaterial, involved in carrying or transporting the subject compound fromone organ, or portion of the body, to another organ, or portion of thebody. Each carrier must be “acceptable” in the sense of being compatiblewith the other ingredients of the formulation and not injurious to thepatient.

As used herein, the term “solution” includes a pharmaceuticallyacceptable carrier or diluent in which the MSCs used in the inventionremain viable.

The term “substantially pure”, with respect to MSC populations, refersto a population of cells in which at least about 50%, 60%, 70%, 75%, atleast about 85%, at least about 90%, or at least about 95%, by number ofthe cells are MSCs. In other words, the term “substantially pure”, withrespect to MSC populations, refers to a population of cells thatcontains less than about 50%, less than about 30%, less than about 20%,less than about 10%, or less than about 5%, by number of lineagecommitted cells. “Support” as used herein refers to any device ormaterial that may serve as a foundation or matrix for the growth ofadipose tissue-derived stromal cells.

DESCRIPTION

A first aspect of the invention refers to a composition comprising aconditioned cell culture medium obtained or obtainable by a processwhich comprises culturing a population of mesenchymal stromal cells(MSCs), in which at least about 25%, at least about 30%, at least about35%, at least about 40%, at least about 45%, at least about 50%, atleast about 55%, at least about 60%, at least about 65%, at least about70%, at least about 75%, at least about 80%, at least about 85%, atleast about 90%, at least about 95%, at least about 96%, at least about97%, at least about 98%, or at least about 99%, of said population bynumber of cells are MSCs obtained from a mammal pertaining to the genusCanis, in a nutrient rich liquid prepared for cell culture, preferably abasal media; and collecting the conditioned cell culture medium,

wherein preferably the nutrient rich liquid is an animal and humanserum-free media designed to grow MSCs, and wherein said compositionpreferably does not comprise dimethyl sulfoxide (DMSO),for use in therapy.

The MSCs of the first aspect of the invention are undifferentiatedstromal cells having the capacity to differentiate to other cells, andare typically derived from connective tissue, and are thusnon-hematopoietic cells. The term “connective tissue” refers to tissuederived from mesenchyme and includes several tissues which arecharacterized in that their cells are included within the extracellularmatrix. Among the different types of connective tissues, adipose andcartilaginous tissues are included. In one embodiment, the MSCs are fromthe stromal fraction of the adipose tissue. In another embodiment, theMSCs are obtained from chondrocytes e.g. from hyaline cartilage. Inanother embodiment, the MSCs are obtained from skin. Also, in anotherembodiment, the MSCs are obtained from bone marrow. Alternative sourcesof MSCs include but are not limited to periosteum, dental pulp, spleen,pancreas, ligament, tendon, skeletal muscle, umbilical cord andplacenta. Other sources are induced pluripotent stem cells or embryonicstem cells.

It is important to note that the MSCs used to obtain the composition ofthe first aspect of the invention are obtained from a mammal pertainingto the genus Canis, in particular these cells are canine MSCs.Typically, the canine MSCs are obtained from the stromal fraction ofadipose tissue, i.e. they are canine adipose tissue-derived stromalcells (ASCs) or immortalized MSCs obtained therefrom. The canine MSCsare adherent to plastic under standard culture conditions. Canine M SCsare undifferentiated multipotent cells, having the capacity todifferentiate into or towards somatic cells such as mesodermal cells(e.g. adipose, chondrocytes, osteoblasts) and optionally into or towardsendodermal and/or ectodermal cell types or lineages. Typically, thecells have the capacity to differentiate into or towards at least two orall cell types selected from the group consisting of adipocytic,chondroblastic and osteoblastic lineages.

In one embodiment of the first aspect of the invention, the canine MSCsare in vitro culture expanded canine MSCs or the in vitro cultureexpanded progeny thereof (hereinafter both are referred to as expandedMSCs or “eMSCs”). Methods for the preparation of eMSCs are known in theart, for example as described in WO2007039150. eMSCs retain severalphenotypic characteristics of MSC, e.g. the eMSCs are adherent toplastic under standard culture conditions and retain an undifferentiatedphenotype.

eMSCs are undifferentiated multipotent cells, having the capacity todifferentiate into somatic cells such as mesodermal cells. Whereas M SCshave the capacity to differentiate towards at least one or morespecialized cell lineages such as but not limited to adipocyte,chondroblastic and osteoblastic lineages; typically in eMSCs thiscapacity to differentiate is reduced or may even be absent e.g. whereasa MSCs may differentiate towards at least the osteogenic and adipocyticlineages, the eMSCs derived therefrom may differentiate only towards theadipocytic lineage. This may be advantageous for therapeuticapplications of the cells, where the cells may be administered topatients as it can reasonably be expected that unanticipated andpotentially harmful differentiation of eMSCs will be less likely tooccur.

In one embodiment the canine eMSCs may be the progeny of stem cells.Typically the canine eMSCs (i) do not express markers specific fromAPCs; (ii) do not express IDO constitutively (iii) do not significantlyexpress MHC II constitutively. Typically expression of IDO or MHC II maybe induced by stimulation with IFN-γ.

In addition, it is further noted that the cell population referred to inthe first aspect of the invention is also referred to as “cellpopulations of the invention”. Typically, the canine MSCs are expandedASCs, typically allogeneic or autologous expanded ASCs. Typically, thecell populations of the invention are a homogenous or substantiallyhomogenous population of canine MSCs and/or canine eMSCs. Cellpopulations of the invention comprise essentially canine MSCs and/orcanine eMSCs, however cell populations of the invention may alsocomprise other cell types. Accordingly in one embodiment the inventionprovides cell populations of the invention in which at least about 25%,at least about 30%, at least about 35%, at least about 40%, at leastabout 45%, at least about 50%, at least about 55%, at least about 60%,at least about 65%, at least about 70%, at least about 75%, at leastabout 80%, at least about 85%, at least about 90%, at least about 95%,at least about 96%, at least about 97%, at least about 98%, or at leastabout 99%, of the cells are canine MSCs and/or canine eMSCs.

In one embodiment, the eMSCs are eASCs, for example human eASCs. In aparticular embodiment, the cells are allogeneic with respect to thesubject to be treated. In another particular embodiment, the cells areautologous with respect to the subject to be treated.

Typically, a cell population of the invention may have the capacity todifferentiate towards at least one or more specialized cell lineagessuch as but not limited to adipocytic, chondroblastic and osteoblasticlineages. In one embodiment a cell population of the invention may havethe capacity to differentiate into or towards at least two or all celltypes selected from the group consisting of adipocytic, chondroblasticand osteoblastic lineages. However, in an alternative embodiment thiscapacity to differentiate is reduced or may even be absent e.g. whereasa MSC may differentiate towards at least the osteogenic and adipocyticlineages, the eMSC population derived therefrom may differentiate onlytowards the adipocytic lineage. This may be advantageous for therapeuticapplications of the cells, where the cells may be administered topatients as it can reasonably be expected that unanticipated andpotentially harmful differentiation of eMSCs will be less likely tooccur.

As refer in the first aspect of the invention, and as exemplified in theexamples, we provide a medium which is conditioned by culture ofmesenchymal stem cells (MSCs) obtained from a mammal pertaining to thegenus Canis. Such a conditioned medium comprises molecules secreted bysuch MSCs, including unique gene products. Such a conditioned medium,and combinations of any of the molecules comprised therein, including inparticular proteins or polypeptides, may be used in the treatment of adisease. They may be used to supplement the activity of, or in place of,the MSCs, for the purpose of for example treating or preventing adisease.

The conditioned medium of the first aspect of the invention may be madeby culturing canine MSCs in a medium, such as a nutrient rich liquidprepared for cell culture, preferably a basal media; wherein thenutrient rich liquid is preferably an animal and human serum-free mediadesigned to grow MSCs, for a predetermined length of time. Theconditioned medium will thus comprise polypeptides secreted by thecanine MSCs, as described in the Examples.

In a preferred embodiment, the serum-free media comprises Knockout DMEMmedia (Invitrogen-Gibco, Grand Island, N.Y.).

Notwithstanding the above, the conditioned media of the first aspect ofthe invention may be made by any media suitable for propagating canineMSCs and can thus have any of several different formulas, as long as thecells obtained have the desired characteristics, and can be propagatedfurther. Suitable sources are as follows: Dulbecco's modified Eaglesmedium (DMEM), Gibco #I 1965-092; Knockout Dulbecco's modified Eaglesmedium (KO DMEM), Gibco #10829-018; 200 mM L-glutamine, Gibco#15039-027; non-essential amino acid solution, Gibco 11140-050;beta-mercaptoethanol, Sigma #M7522; human recombinant basic fibroblastgrowth factor (bFGF), Gibco #13256-029. Exemplary serum-containingembryonic stem (ES) medium is made with 80% DMEM (typically KO DMEM),20% defined fetal bovine serum (FBS) not heat inactivated, 0.1 mMnon-essential amino acids, 1 mM L-glutamine, and 0.1 niMbeta-mercaptoethanol. The medium is filtered and stored at 4 degrees C.for no longer than 2 weeks. Serum-free embryonic stem (ES) medium ismade with 80% KO DMEM, 20% serum replacement, 0.1 mM non-essential aminoacids, 1 mM L-glutamine, and 0.1 mM beta-mercaptoethanol. An effectiveserum replacement is Gibco #10828-028. The medium is filtered and storedat 4 degrees C. for no longer than 2 weeks. Just before use, human bFGFis added to a final concentration of 4 ng/niL (Bodnar et al., GeronCorp, International Patent Publication WO 99/20741).

In a preferred embodiment, the media comprises Knockout DMEM media(Invitrogen-Gibco, Grand Island, N.Y.), supplemented with 10% serumreplacement media (Invitrogen-Gibco, Grand Island, N.Y.), 5 ng/ml FGF2(Invitrogen-Gibco, Grand Island, N.Y.) and 5 ng/ml PDGF AB (Peprotech,Rocky Hill, N.J.).

The conditions for culturing the canine MSCs in order to obtain theconditioned media of the present invention are clearly illustrated inexample 1; however, these conditions can be optimized appropriately.

The conditioned medium of the present invention may be used in therapyas is, or after one or more treatment steps. For example, theconditioned medium may be UV treated, filter sterilised, etc. One ormore purification steps may be employed.

In particular, the conditioned media may be concentrated, for example bydialysis or ultrafiltration. For example, the medium may be concentratedusing membrane ultrafiltration with a nominal molecular weight limit(NMWL) of for example 3K.

We also provide for a composition comprising one or more, preferablyall, of the polypeptides described in the Examples, in lieu of, or tosupplement such a conditioned medium.

Conditioned media from mesenchymal stem cells such as those madeaccording to the methods and compositions described here may be used fora variety of commercially important research, diagnostic, andtherapeutic purposes.

The conditioned media from mesenchymal stem cells may in particular beused for the preparation of a pharmaceutical composition for thetreatment of disease. Such disease may comprise a disease treatable byregenerative therapy, including skin disease, burns, psoriasis or atopicdermatitis. In this sense, it is herein noted that, as shown in theexamples, analysis of the secreted proteins to the culture media by thecanine MSCs (see table II) shows that the proteins expressed areinvolved in a number of biological processes including: tissuedifferentiation including vascularization, hematopoiesis and skeletaldevelopment. We therefore provide generally for the use of mesenchymalstem cells (MSCs) obtained from a mammal pertaining to the genus Canisor medium conditioned by mesenchymal stem cells (MSCs) obtained from amammal pertaining to the genus Canis, in the regulation of any of thesebiological processes.

Furthermore, the mesenchymal stem cells (MSCs) obtained from a mammalpertaining to the genus Canis may be used to treat diseases in whichthese biological processes may have a role in, or whose repair ortreatment involves any one or more of these biological processes.Similarly, the proteins expressed by the mesenchymal stem cells (MSCs)obtained from a mammal pertaining to the genus Canis, singly or incombination, preferably in the form of a conditioned medium, may be usedto supplement the activity of, or in place of, the mesenchymal stemcells (MSCs) obtained from a mammal pertaining to the genus Canis, forthe purpose of for example treating or preventing such diseases.

Accordingly, such a conditioned medium may be used to treat a cutaneouswound, a dermatologic disorder, a dermatological lesion, dermatitis,psoriasis, condyloma, verruca, hemangioma, keloid, skin cancer, atopicdermatitis, Behcet disease, chronic granulomatous disease, cutaneous Tcell lymphoma, and ulceration. In particular, such a conditioned mediummay be used to treat psoriasis or atopic dermatitis. In addition, theconditioned medium may be used to aid wound healing or scar reduction inan individual.

In particular, the conditioned medium may be used to regulate theprocesses involved in vascularisation, hematology (specifically immuneprocesses) or musculoskeletal development, etc.

Preferably, said conditioned medium or a composition containing orcomprising the same, does not comprise dimethyl sulfoxide (DMSO), and isused in the xenogeneic treatment of atopic dermatitis, eczema and/orpsoriasis in a human subject.

Furthermore, any one or more proteins secreted from the MSCs describedhere, including in the form of conditioned media, may be used for thesame purposes as the MSCs described herein. We therefore provide acomposition comprising one or more, preferably substantially all, thepolypeptides which are present in the conditioned medium. Specifically,we provide a composition comprising one or more, preferablysubstantially all, the polypeptides set out in table II, in particularof the case of MSCs derived from the adipose tissue of a dog.

Such a composition may be used for any purpose the conditioned mediummay be used. Unless the context dictates otherwise, the term“conditioned medium” should be taken to include not only cell culturemedium exposed to MSCs as well as such a composition comprising one ormore, preferably substantially all, the polypeptides which are presentin the conditioned medium (see table II).

It will be evident that the methods and compositions described hereenable the production of conditioned media from mesenchymal stem cells.Thus, any uses of mesenchymal stem cells will equally attach toconditioned media from mesenchymal stem cells.

The conditioned medium as described in this document may be delivered tothe human or animal body by any suitable means.

We therefore describe a pharmaceutical or cosmetic composition ordelivery system for delivering a conditioned medium as described in thisdocument to a target cell, tissue, organ, animal body or human body, andmethods for using the pharmaceutical or cosmetic composition or deliverysystem to deliver conditioned medium to a target. The delivery systemmay comprise a source of conditioned medium, such as a containercontaining the conditioned medium. The delivery system may comprise adispenser for dispensing the conditioned medium to a target.

Accordingly, we provide a delivery system for delivering a conditionedmedium, comprising a source of conditioned medium as described in thisdocument together with a dispenser operable to deliver the conditionedmedium to a target.

We further provide for the use of such a delivery system in a method ofdelivering a conditioned medium to a target.

Delivery systems for delivering fluid into the body are known in theart, and include injection, surgical drips, cathethers (includingperfusion cathethers) such as those described in U.S. Pat. No.6,139,524, for example, drug delivery catheters such as those describedin U.S. Pat. No. 7,122,019.

It will be evident that the particular delivery should be configurableto deliver the required amount of conditioned medium at the appropriateinterval, in order to achieve optimal treatment. A preferred delivery oradministration of the conditioned medium is the topical delivery oradministration. For this purpose, the composition or delivery system mayhave excipients. An excipient as defined in present specification, is aninactive substance used as a carrier or vehicle for the conditionedmedium comprised in the composition. The compositions of the inventionmay comprise any excipient, preferably suitable for being included intopical compositions (i.e. dermatological acceptable excipient). Thefollowing auxiliary agents mentioned below can independent of each otherbe present in the composition of the invention: gelling agents, oils,waxes, thickening agents, hydrophilic or hydrophobic polymers,emulsifying agents, emollients, fatty acids, organic solvents,antioxidants, stabilizers, sequestering agents, acidifying or basifyingagents, emulsifiers, emollients, surfactants, film formers, biologicaladditives to enhance performance and/or consumer appeal such as aminoacids, proteins, vanilla, aloe extract or bioflavinoids, bufferingagents, chelating agents such as ethylenediaminetetra-acetic acid (EDTA)or oxalic acid, colorants, dyes, propellants, antifoaming agents,wetting agents, vitamins, emulsion stabilizers, pH adjusters, thickeningagents, fragrances, preservatives, opacifying agents, water and/oralcohols. The aforementioned auxiliary agents for the composition of theinvention are used in the usual amounts known by those skilled in theart. Suitable oils for the compositions of the invention are selectedfrom animal or vegetable or synthetic oils. Particularly preferred oilsare selected from the group comprising liquid petrolatum, liquidparaffin, volatile and non-volatile silicone oils, isoparaffins,polyalphaolefins, fluorated and perfluorated oils. Suitable stabilizersfor the compositions of the invention can be of non-ionic, anionic,cationic and amphiphilic nature. Preferred stabilizers are selected fromthe group comprising polyethylenglycol (PEG) and derivatives thereof,tweens, tritons, spans, polygycerines, polyalkyl glycerides, alkylsulfonates, aryl sulfonates, alkyl phosphates, derivatives ofalkyl-betaine and phosphatidylglycerole.

Emulsifiers may be used in the compositions of present invention inamounts effective to provide uniform blending of ingredients of saidcompositions. Suitable emulsifiers include anionics such as fatty acidsoaps, e.g., potassium stearate, sodium stearate, ammonium stearate, andtriethanolamine stearate; polyol fatty acid monoesters containing fattyacid soaps, e.g., glycerol monostearate containing either potassium orsodium salt; sulfuric esters (sodium salts), e.g., sodium lauryl 5sulfate, and sodium acetyl sulfate; and polyol fatty acid monoesterscontaining sulfuric esters, e.g., glyceryl monostearate containingsodium lauryl surfate; (ii) cationics chloride such as N (stearoylcolamino formylmethyl) pyridium; N-soya-N-ethyl morpholiniumethosulfate; alkyl dimethyl benzyl ammonium chloride;diisobutylphenoxytheoxyethyl dimethyl benzyl ammonium chloride; andacetyl pyridium chloride; and (iii) nonionics such as polyoxy ethylenefatty alcohol ethers, e.g., monostearate; polyoxy ethylene laurylalcohol; polyoxypropylene fatty alcohol ethers, e.g., propoxylated oleylalcohol; polyoxyethylene fatty acid esters, e.g., polyoxyethylenestearate; polyoxyethylene sorbitan fatty acid esters, e.g.,polyoxyethylene sorbitan monostearate; sorbitan fatty acid esters, e.g., sorbitan; polyoxyethylene glycol fatty acid esters, e.g.,polyoxyethylene glycol monostearate; and polyol fatty acid esters, e.g.,glyceryl monostearate and propylene glycol monostearate; and ethoxylatedlanolin derivatives, e.g., ethoxylated lanolins, ethoxylated lanolinalcohols and/or ethoxylated cholesterol.

Emollients may be also used in the compositions of the invention in suchamounts to prevent or relieve dryness. Suitable emollients include,without limitation hydrocarbon oils and waxes; silicone oils;triglyceride esters; acetoglyceride esters; ethoxylated glyceride; alkylesters; alkenyl esters; fatty acids; fatty alcohols; fatty alcoholethers; etheresters; lanolin and derivatives; polyhydric alcohols(polyols) and polyether derivatives; polyhydric alcohol (polyol) esters;wax esters; beeswax derivatives; vegetable waxes; phospholipids;sterols; and/or amides.

Surfactants can further be used too in the compositions of presentinvention. Suitable surfactants are for example those surfactantsgenerally grouped as cleansing agents, emulsifying agents, foamboosters, hydrotropes, solubilizing agents, suspending agents andnon-surfactants, which facilitate the dispersion of solids in liquids.

Film formers which may be preferably used in the compositions of presentinvention should keep the composition smooth and even and arepreferably, without limitation. Suitable film formers are selected fromthe group comprising acrylamide/sodium acrylate copolymer; ammoniumacrylates copolymer; Balsam Peru; cellulose gum; ethylene/maleicanhydride copolymer; hydroxyethylcellulose; hydroxypropylcellulose;polyacrylamide; polyethylene; polyvinyl alcohol; pvm/MA copolymer (vinylmethyl ether/maleic anhydride copolymer); PVP (polyvinylpyrrolidone);maleic anhydride polymer, vinylpyrrolidon/hexadecene copolymer;acryliclacrylate copolymer and the like. PH adjusters may also be usedin the compositions of present invention. These pH adjusters arepreferably selected from: ammonium hydroxide, triethanolamine or citricacid.

Thickening agents used for the compositions of the invention preferablyare selected from: candelilla, carnauba, and microcrystalline waxes,crosslinked acrylic-acid polymers, carbomer,methylhydroxyethylcellulose, hydroxypropylmethylcellulose or hydroxyethylcellulose and polyethylene thickeners.

Examples of preferred organic solvents for the compositions of presentinvention include lower aliphatic alcohols and polyols.

Suitable antioxidants for the compositions used in present invention arepreferably selected from the group comprising ascorbic acid (vitamin C),sodium-L-ascorbate, calcium-L-ascorbate, ascorbyl palmitate,butylhydroxyanisole, butylhydroxytoluene, calcium-disodium-EDTA,isoascorbic acid, lecitine, lactic acid, polyphosphate, tocopherol(vitamin E), like [alphaj-tocopherol, [gamma]-tocopherol,[delta]-tocopherol, propylgallate, octylgallate, dodecylgallate,sodium-isoascorbate, citric acid, sodium citrate, potassium citrate andtin-II-chloride. Gelling agents, which are preferably used in thecompositions of the invention, can be natural or synthetic polymers.Natural polymers are preferably selected from: Agar-Agar, alginate,pectin, carbomer, carrageenan, casein, dextrine, gelatine, arabic gum,keratine, locust bean gum, xanthan gum and the like. Preferred syntheticpolymers which can be used in the compositions of the invention areselected from: acylic acid polymers, polyacryl amides and alkylene oxidepolymers.

As already indicated, any route of administration can be used foradministering the compositions of the invention to the subject, beingthe preferred routes of administration intravenous, oral and topical. Ina preferred embodiment the composition of the invention is a topicalformulation that may be formulated in liquid or in semi-solid form,preferably as liquid, fluid, foam, cream, gel, paste, balsam, spray,ointment, lotion, conditioner, tonic, milk, mousse, emulsion, serum,oil, stick, shampoo, jelly, suspension, dispersion, lacquer, paint,elixir, drop or aerosol. In a particular embodiment, the activecompounds of the invention were administered topically in a liposomalpreparation. As used in the present invention, a “liposome” is anartificial vesicle that is composed of one or more concentric layers andis used especially to deliver substances (i.e. conditioned medium) tothe body cells.

Topical administration of the composition of the invention leads to ahigh bioavailability of the active compounds of the conditioned medium.In pharmacology, bioavailability is used to describe the fraction of anadministered dose of unchanged drug that reaches the systemiccirculation, one of the principal pharmacokinetic properties of drugs.Therefore, the bioavailability is one of the essential tools inpharmacokinetics, and it must be considered when calculating dosages fornon-intravenous routes of administration.

It will be evident that the delivery method will depend on theparticular organ to which the conditioned medium is to be delivered, andthe skilled person will be able to determine which means to employaccordingly.

The following examples are merely for illustrative purposes and do notlimit the present invention.

EXAMPLES Example 1. Obtention of the Conditioned Media of the PresentInvention

In general terms the conditioned media of the present invention can beobtained by culturing MSCs (mesenchymal stem cells), preferably derivedfrom dog adipose tissue, in a suitable cell media, preferably in DMEMsupplemented with 10% FBS and PSG, until reaching a confluence ofbetween 70% and 80%, such level of confluence is usually reached inpassage 3.

The adipose tissue is obtained from one or several healthy donors. About10-500 g of adipose tissue from each donor will produce one batch ofmaster cell banks. The age of the donor may influence the in vitrolifespan of the cells, so the original donors will be under two years ofage. The clinical history and the good general conditions of the donorwill be assessed. Several samples will be collected to perform routineand disease tests and certify the absence of pathogens. The extractionof adipose tissue will be performed in an operation room or in a stableset up for the extraction according veterinarian surgeon's criteria. Thetissue extraction will be performed by a qualified vet under animalwelfare conditions.

The region for adipose tissue extraction will be preferably the hepaticfalciform ligament, but adipose tissue can be also obtained from otherintra-peritoneal fat or subcutaneous (ie. dock region). The procedurefor adipose tissue collection will be as follows: Briefly, the zone willbe thoroughly cleaned, shaved and disinfected according standardsurgical techniques. Then, an incision is performed under local orsystemic anaesthesia. The extracted adipose tissue is placed in asterile recipient and processed accordingly.

Once such level of confluence is reached, the media is then discardedand the cells should be rinsed, preferably three times, with a salinesolution such as PBS. Cells should be then cultured in a serum-free andantibiotic-free medium, such as DMEM, for at least about 24 hours. Theconditioned media should be then collected and filtered through a 0.2-mmfilter to remove cellular debris.

In particular, the conditioned media used in the present examples wasobtained as follows:

Adipose tissue was obtained from dogs (Canis lupus familiaris L.), cats(Felis silvestris catus L.) and horses (Equus caballus L.) and also fromhumans, by aseptically collecting said tissues from the abdominal regionof the animals and humans by surgical excision. MSCs obtained from suchadipose tissue were then culture in DMEM supplemented with 10% FBS andPSG until reaching a confluence of between 70% and 80%, such level ofconfluence was reached in passage 3. The medium was then discarded andthe cells were rinsed three times with PBS. Cells were then culturedwith serum-free and antibiotic-free medium for periods of time rangingfrom 24 to 72 hours. The conditioned media was then collected andfiltered through a 0.2-mm filter to remove cellular debris. It is notedthat such 4 conditioned media (obtained from dogs, horses, humans andcats) was characterized as illustrated in example 2 below.

Cells were detached and characterized as MSC by means of surface markersidentified by flow cytometry.

Example 2. Characterization of the Conditioned Media Obtained in Example1

A Proteome Profiler Array was employed for the characterization of theconditioned media.

Human XL Cytokine Array Kit R&D Systems (Catalog Number ARY022B) wasemployed following manufacturer instructions. It is a rapid andsensitive tool to simultaneously detect cytokine differences betweensamples. This kit can use to measure the relative expression levels ofup to 105 soluble human proteins.

In order to perform the characterization of the conditioned media,capture and control antibodies were spotted in duplicate onnitrocellulose membranes. Cell culture supernatants as obtained inexample 1, were directly incubated overnight with the Proteome ProfilerHuman XL Cytokine Array. The membranes were washed to remove unboundmaterial followed by incubation with a cocktail of biotinylateddetection antibodies. Streptavidin-HRP and chemiluminescent detectionreagents were then applied, and a signal was produced at each capturespot corresponding to the amount of protein bound.

A list of the 105 different detected proteins are shown in Table I.Results for the four-different species can be found in the followingfigures: FIG. 1 (Human), FIG. 2 (Dog), FIG. 3 (Cat) and FIG. 4 (Horse).

TABLE I Human XL Cytokine Array coordinates, showing positive andnegative controls, the analytes checked, the Entrez gene ID of theanalyte, and alternate nomenclature if exists. Entrez CoordinateAnalyte/Control Gene ID Alternate Nomenclature A1, A2 Reference SpotsN/A RS A3, A4 Adiponectin 9370 Acrp30 A5, A6 Apolipoprotein A-I 335ApoA1 A7, A8 Angiogenin 283 — A9, A10 Angiopoietin-1 284 Ang-1, ANGPT1A11, A12 Angiopoietin-2 285 Ang-2, ANGPT2 A13, A14 BAFF 10673 BLyS,TNFSF13B A15, A16 BDNF 627 Brain-derived Neurotrophic Factor A17, A18Complement 727 C5/C5a A19, A20 CD14 929 — A21, A22 CD30 943 TNFRSF8 A23,A24 Reference Spots N/A RS B3, B4 CD40 ligand 959 CD40L, TNFSF5, CD154,TRAP B5, B6 Chitinase 3-like 1 1116 CHI3L1, YKL-40 B7, B8 ComplementFactor D 1675 Adipsin, CFD B9, B10 C-Reactive Protein 1401 CRP B11, B12Cripto-1 6997 Teratocarcinoma- derived Growth B13, B14 Cystatin C 1471CST3, ARMD11 B15, B16 Dkk-1 22943 Dickkopf-1 B17, B18 DPPIV 1803 CD26,DPP4, Dipeptidyl- peptidase B19, B20 EGF 1950 Epidermal Growth FactorB21, B22 Emmprin 682 CD147, Basigin C3, C4 ENA-78 6374 CXCL5 C5, C6Endoglin 2022 CD105, ENG C7, C8 Fas Ligand 356 TNFSF6, CD178, CD95L C9,C10 FGF basic 2247 FGF-2 C11, C12 FGF-7 2252 KGF C13, C14 FGF-19 9965 —C15, C16 Flt-3 Ligand 2323 FLT3LG C17, C18 G-CSF 1440 CSF3 C19, C20GDF-15 9518 MIC-1 C21, C22 GM-CSF 1437 CSF2 D1, D2 GROα 2919 CXCL1,MSGA-α D3, D4 Growth Hormone 2688 GH, Somatotropin D5, D6 HGF 3082Scatter Factor, SF D7, D8 ICAM-1 3383 CD54 D9, D10 IFN-γ 3458 IFNG D11,D12 IGFBP-2 3485 — D13, D14 IGFBP-3 3486 — D15, D16 IL-1α 3552 IL-1F1D17, D18 IL-1β 3553 IL-1F2 D19, D20 IL-1ra 3557 IL-1F3 D21, D22 IL-23558 — D23, D24 IL-3 3562 — E1, E2 IL-4 3565 — E3, E4 IL-5 3567 — E5, E6IL-6 3569 — E7, E8 IL-8 3576 CXCL8 E9, E10 IL-10 3586 — E11, E12 IL-113589 — E13, E14 IL-12 p70 3593 — E15, E16 IL-13 3596 — E17, E18 IL-153600 — E19, E20 IL-16 3603 — E21, E22 IL-17A 3605 IL-17, CTLA8 E23, E24IL-18 Bpa 10068 — F1, F2 IL-19 29949 — F3, F4 IL-22 50616 IL-TIF F5, F6IL-23 51561 IL-23A, SGRF F7, F8 IL-24 3627 C49A, FISP, MDA-7, MOB-5,ST16 F9, F10 IL-27 246778 — F11, F12 IL-31 386653 — F13, F14 IL-32 9235— F15, F16 IL-33 90865 C9orf26, DVS27, NF-HEV F17, F18 IL-34 146433C16orf77 F19, F20 IP-10 3627 CXCL10 F21, F22 I-TAC 6373 CXCL11, SCYB9BF23, F24 Kallikrein 3 354 PSA, KLK3 G1, G2 Leptin 3952 OB G3, G4 LIF3976 — G5, G6 Lipocalin-2 3934 NGAL, LCN2, Siderocalin G7, G8 MCP-1 6347CCL2, MCAF G9, G10 MCP-3 6354 CCL7, MARC G11, G12 M-CSF 1435 CSF1 G13,G14 MIF 4282 — G15, G16 MIG 4283 CXCL9 G17, G18 MIP-1α/MIP-1β 6348/6351CCL3/CCL4 G19, G20 MIP-3α 6364 CCL20, Exodus-1, LARC G21, G22 MIP-3β6363 CCL19, ELC G23, G24 MMP-9 4318 CLG4B, Gelatinase B H1, H2Myeloperoxidase 4353 MPO, Lactoperoxidase H3, H4 Osteopontin 6696 OPNH5, H6 PDGF-AA 5154 — H7, H8 PDGF-AB/BB 5154/5155 — H9, H10 Pentraxin 35806 PTX3, TSG-14 H11, H12 PF4 5196 CXCL4 H13, H14 RAGE 177 — H15, H16RANTES 6352 CCL5 H17, H18 RBP-4 5950 — H19, H20 Relaxin-2 6019 RLN2,RLXH2 H21, H22 Resistin 56729 ADSF, FIZZ3, RETN H23, H24 SDF-1α 6387CXCL12, PBSF I1, I2 Serpin E1 5054 PAI-I, PAI-1, Nexin I3, I4 SHBG 6462ABP I5, I6 ST2 9173 IL-1 R4, IL1RL1, ST2L I7, I8 TARC 6361 CCL17 I9, I10TFF3 7033 ITF, TFI I11, I12 TfR 7037 CD71, TFR1, TFRC, TRFR I13, I14TGF-α 7039 TGFA I15, I16 Thrombospondin-1 7057 THBS1, TSP-1 I17, I18TNF-α 7124 TNFSF1A I19, I20 uPAR 5329 PLAUR I21, I22 VEGF 7422 BEGFA J1,J2 Reference Spots N/A RS J5, J6 Vitamin D BP 2638 VDB, DBP, VDBP J7, J8CD31 5175 PECAM-1 J9, J10 TIM-3 84868 HAVCR2 J11, J12 VCAM-1 7412 CD106J23, J24 Negative Controls N/A Control (−)

Comparative results of the mean pixel density can be found in table II.

TABLE II Mean pixel density of representative blots from the fouranalysed species. MEAN PIXEL DENSITY Coordinate Analyte Human Dog CatHorse A7, A8 Angiogenin 16843 0 0 86 B5, B6 Chitinase 3-like 1 22816 0521 13 B7, B8 Complement Factor 11286 74 0 95 B21, B22 Emmprin 6109 1775431 1055 C5, C6 Endoglin 13619 0 335 0 C11, C12 FGF-7 11032 0 20 0 C13,C14 FGF-19 5339 170 284 343 C19, C20 GDF-15 13684 142 133 78 D11, D12IGFBP-2 17762 12412 3225 10783 D13, D14 IGFBP-3 8568 0 71 4 E7, E8 IL-88153 0 227 37 E21, E22 IL-17A 4800 311 709 870 G7, G8 MCP-1 20066 37 7682006 G13, G14 MIF 15445 0 4358 159 G23, G24 MMP-9 3980 400 373 514 H5,H6 PDGF-AA 1731 150 3482 65 H9, H10 Pentraxin 3 10306 8 555 85 H23, H24SDF-1α 6486 1146 609 629 I1, I2 Serpin E1 26138 6871 2264 837 I15, I16Thrombospondin-1 18335 14541 17516 49 I21, I22 VEGF 10296 10239 1095 941

In view of these results, it is thus clear that MSCs from differentorigins provide different conditioned media under a qualitative andquantitative view point.

The inventors of the present invention have surprisingly found thatconditioned media obtained from dogs is particularly effective for thetreatment of skin diseases such as atopic dermatitis and psoriasis notonly in animals but also in human beings. The success in the xenogeneicuse from dogs to humans, is not obvious. Moreover, the success is notobvious given the high phylogenetic divergence that exist between thetwo species.

Example 3. Clinical and Veterinary Results with the Composition of theInvention

1^(st) Case. Treatment of Atopic Dermatitis in a Dog

An eight years old Pekingese with a cutaneous disease, an atopicdermatitis that caused a testicular affectation and a lot of itching wastaken as a candidate for the treatment of the invention. In thisindividual, response to the usual corticosteroid prescription relapsedfrequently. We treated such cutaneous disease employing the dogconditioned media as a cutaneal lotion over the wounds, spreading it twotimes a day during 6 weeks.

One and a half months after treatment with conditioned media, Pancho isin complete remission as illustrated in FIG. 5.

2^(nd) Case. Treatment of a Siamese Cat Who Suffered from Severe SkinBurn Injuries.

Five years ago the Siamese cat subject was dropped into a frying pan ofboiling oil. 30% of the body was affected, mainly legs and one of theflanks. Initial treatment consisted on silver sulfadiazid, ointment andliquid.

The cat recovered of the lesions during the first year, but a zone ofapproximately 10×5 cm was not recovered producing stable lesions overthe last 3 years, with continuous exudate and itching.

We treated such stable lesions employing the dog conditioned media as acutaneal lotion over the wound, spreading it two times a day during 6weeks

One and a half months after treatment with conditioned media, the sizeof the wound decreased as illustrated in FIG. 6.

3^(rd) Case. Treatment of Psoriasis in a Human being

Male. 79 years old. Diagnosed of Psoriasis in 1975. The affected areaswere the elbows and the right leg, where a large spot from the kneealmost to the ankle could be clearly seen. For about eight or ten yearssmall affected areas in hands.

The human subject suffered numerous treatments, including corticosteroidtreatments.

We treated such stable lesions with dog conditioned media during 35days, two times per day.

After employment of the conditioned culture, applied directly on theskin lesions in legs and hands employing a vaporizer, peeling and scalesdisappeared from the skin.

The small affected parts of the hands almost disappeared completely.Please refer to FIG. 7.

4^(rd) Case. Treatment of Psoriasis in a Human being

Female. 37 years old. Diagnosed of Psoriasis Arthritis for more than 10years.

Treatment with salazopyrine (4 tablets/day). For acute crises, thetreatment consisted of small doses of methotrexate. Such treatment wasconsidered inefficient.

We treated the lesions produced by the psoriasis with dog conditionedmedia.

After 21 days employing the conditioned media, skin peeling and scalesdiminished notably as shown in FIG. 8.

5^(rd) Case. Treatment of Psoriasis in a Human being

58 year old male. Diagnosed of psoriasis in the spring of 1980, withaffected areas located in elbows.

Treatment of cream steroids in areas of major relevance withoutsignificant improvement.

Change to treatment with petroleum/iodine preparation without positiveeffects (1981).

Change to Use of Acetylsalicylic Acid for years without furtheraffecting scaling. (1982)

Punctual appearance on the scalps that appeared and disappeared andnever in a critical or annoying state. (1990- . . . )

Abandonment of Acetylsalicylic acid and use of a moisturizer in areas ofelbows, hands (1996-present).

We treated the lesions produced by the psoriasis for with dogconditioned media.

After 36 days employing the conditioned media, skin peeling and scalesdiminished notably as shown in FIG. 9.

1. A method of xenogenically treating psoriasis in a human subject, themethod comprising administering to the human subject having psoriasis anamount of a composition comprising a conditioned cell culture mediumeffective for xenogenic treatment of psoriasis in the human subject, theconditioned cell culture medium made by a process comprising: culturinga population of mesenchymal stromal cells (MSCs) or immortalized cellsobtained therefrom, in which at least 50% of said population by numberof cells are MSCs obtained from a mammal of the genus Canis orimmortalized cells obtained therefrom, in a nutrient rich liquid or abasal media suitable for propagating the MSCs; and collecting theconditioned cell culture medium,
 2. The method according to claim 1,wherein the MSCs are obtained from a dog species.
 3. The methodaccording to claim 1, wherein the MSCs are umbilical-cord derivedstromal cells, adipose tissue-derived stromal cells, expandedmesenchymal stromal cells, expanded adipose tissue-derived stromalcells, bone-marrow derived stromal cells, expanded bone-marrow derivedstromal cells, or immortalized mesenchymal stromal cells obtainedtherefrom.
 4. The method according to claim 1, wherein the nutrient richliquid prepared for cell culture is a buffered saline solutioncomprising amino acids and vitamins supplemented with sodium pyruvateand glutamine.
 5. The method of claim 1, wherein the nutrient richliquid is a basal media with supplementation.
 6. The method of claim 1,wherein such composition is formulated to deliver an amount ofconditioned medium at an appropriate interval to effectively treatpsoriasis.
 7. The method according to claim 6, wherein such compositionis formulated for administration by an intravenous, oral, or topicalroute.
 8. The method of claim 7, wherein such route of administration istopical, and wherein the composition is a topical formulation that isformulated in liquid or in semi-solid form.
 9. The method of claim 7,wherein such route of administration is topical, and wherein thecomposition is a topical formulation that is formulated in a formselected from: a liquid, a fluid, a foam, a cream, a gel, a paste, abalsam, a spray, an ointment, a lotion, a conditioner, a tonic, a milk,a mousse, an emulsion, a serum, an oil, a stick, a shampoo, a jelly, asuspension, a dispersion, a lacquer, a paint, an elixir, a drop and anaerosol form.
 10. The method of claim 7, wherein such route ofadministration is topical, and wherein the composition is a topicalformulation that is formulated in a liposomal preparation.